Receptor-mediated phospholipase A2 activation and eicosanoid generation was studied in neural cells in primary culture. Alpha-1 adrenergic, muscarinic acetylcholine, and serotonergic receptors demonstrated brain region specific release of arachidonic acid. The receptor mediated release of arachidonic acid was neural cell in origin suggesting a role for eicosanoids in neural transmission. Mechanistic studies suggested that the release of arachidonic acid was the consequence of phospholipase A2 and not phospholipase C activation, a potential alternate source of free arachidonic acid. The studies of the mechanism of receptor-mediated arachidonic acid release were extended using mammalian fibroblast transfected and stably expressing, muscarinic acetylcholine receptors. It was shown that muscarinic receptors m1, m3, and m5 couple to a sustained calcium influx. In CHO cells, the calcium influx, coincident with protein kinase C activation, appears to regulate phospholipase A2 activity. Using muscarinic receptor chimeras, the calcium influx was attributed to a voltage insensitive calcium channel coupled to the muscarinic receptor through a G protein. Additional studies using transfected fibroblast suggested that many receptors not directly involved in phospholipase A2 stimulation, may play a modulatory role in arachidonic acid release.